This invention relates to plant physiology, growth, development, defense and, in particular, to plant genes, termed galacturonosyltransferases (GALATs), nucleic acids encoding same and the uses therefor.
Pectins are the most complex polysaccharides in the plant cell wall. They comprise 30-40% of the primary wall of dicots and non-graminaceous monocots, and ˜10% of the primary wall in the grass family. Pectins are a family of polysaccharides6,8,27 that include homogalacturonan (HGA) (FIG. 1), rhamnogalacturonan-I (RG-I) (FIG. 2) and rhamnogalacturonan II (RG-II) (FIG. 3) as well as xylogalacturonans (XGA)32,34,38 and apiogalacturonans.6,37 While the specific structure of each of these polysaccharides differs as shown in FIGS. 1-3, they are grouped into one family since they appear to be linked to each other in the wall and they each contain α-D-galacturonic acid connected by a 1,4-linkage.
HGA is the most abundant pectic polysaccharide, accounting for ˜55%-70% of pectin39. HGA is a linear homopolymer of α1,4-linked D-galactosyluronic acid that is partially methylesterified at the C6 carboxyl group and may be partially acetylated at O-2 and/or O-38 (FIG. 1). Some plants also contain HGA that is substituted at the 2 or 3 position by D-apiofuranose, the so-called apiogalacturonans (AGA)36,37 and/or HGA that is substituted at the 3 position with D-xylose32-35, so-called xylogalacturonan (XGA). RG-II is a complex polysaccharide that accounts for approximately 10-11% of pectin8,39. RG-II has an HGA backbone with four structurally complex side chains attached to C-2 and/or C-3 of the GalA8,27 (FIG. 3). Rhamnogalacturonan I (RG-I) accounts for 20-35% of pectin39 (FIG. 2). RG-I is a family of polysaccharides with an alternating [→4)-α-D-GalA-(1→2)-α-L-Rha-(1→] backbone in which roughly 20-80% of the rhamnoses are substituted by arabinan, galactan, or arabinogalactan side branches6,8,30.
Pectins are believed to have multiple roles during plant growth, development, and in plant defense responses. For example, pectic polysaccharides play essential roles in cell wall structure43, cell adhesion44 and cell signaling45,46. Pectins also appear to mediate pollen tube growth47 and to have roles during seed hydration48,49, leaf abscission50, water movement51, and fruit development47,8. Oligosaccharides cleaved from pectin also serve as signals to induce plant defense responses52,53. Studies of mutant plants with altered wall pectin reveal that modifications of pectin structure leads to dwarfed plants43, brittle leaves44, reduced numbers of side shoots and flowers54, malformed stomata44 and reduced cell adhesion55.
Although pectins appear to have multiple roles in plants, in no case has their specific mechanism of action been determined. One way to directly test the biological roles of pectins, and to study their mechanisms of action, is to produce plants with specific alterations in pectin structure. This can be done by knocking out genes that encode the pectin biosynthetic enzymes. Such enzymes include the nucleotide-sugar biosynthetic enzymes and the glycosyltransferases that synthesize the pectic polysaccharides. Each glycosyltransferase is expected to transfer a unique glycosyl residue in a specific linkage onto a specific polymeric/oligomeric acceptor. To date, only five56-59,136 of the more than 200 predicted wall biosynthetic glycosyltransferases have been funtionally identified at the gene level (i.e. enzyme activity of the gene product proven), and none of these have been shown to encode pectin biosynthetic enzymes.
Based on the known structure of pectin, at least 58 distinct glycosyl-, methyl- and acetyl-transferases are believed to be required to synthesize the family of polymers known as pectin. As shown in the review by Mohnen, D. (2002) “Pectins and their Manipulation”, G. B. Seymour et al., Blackwell Publishing and CRC Press, Oxford, England, pp. 52-98, and Table I below, a minimum of 4-9 galacturonosyltranferases are predicted to be required for the synthesis of HGA, RG-I, RG-II and possibly for the synthesis of the modified forms of HGA known as XGA and AGA. The present invention relates to the identification of the first gene, GALAT1, encoding a galacturonosyltranferase and related genes thereto. The studies disclosed hereinbelow led the inventors to conclude that the gene GALAT1 encodes the enzyme known as UDP-GalA:Homogalacturonan α-1,4-Galacturonosyltransferase.
TABLE IList of galacturonosyltransferase activities predicted to be required for pectinbiosynthesis9Type ofWorking1ParentEnzyme3Ref forGalATNumberpolymer2Acceptor substrateEnzyme activityStructureD-GalAT1HGA*GalAα1→4GalAα1,4-GalAT27D-GalAT2RG-IL-Rhaα1→4GalAα1,2-GalAT27-29D-GalAT3RG-IIL-Rhaβ1→3Apifα1,2-GalAT30, 31D-GalAT4RG-IIL-Rhaβ1→3Apifβ1,3GalAT30, 31D-GalAT5?4RG-I/HGAGalAα1→2LRhaα1,4-GalAT—D-GalAT6?RG-II/HGAGalAα1→4GalAα1,4-GalAT—D-GalAT7?XGAGalAα1→4(Xyl β1→3)GalA5α1,4-GalAT32-35D-GalAT8?AGAGalAα1→4(Apif β1→2)GalAα1,4-GalAT36, 37D-GalAT9?AGAGalAα1→4(Apif β1→3)GalAα1,4-GalAT36, 371Numbers for different members of the same groups are given based on pectin structure and on the assumption that HGA is synthesized first, followed by RG-I and RG-II. The numbers were given9 to facilitate a comparison of the enzymes, but final numbering will likely correspond to the order in which the genes are identified.2HGA: homogalacturonan; RG-I: Rhamnogalacturonan I; RG-II: Rhamnogalacturonan II; XGA: Xylogalacturonan; AGA; Apiogalacturonan.3All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor.4The ? means the designated GalAT may be required if a different GalAT in the list does not perform the designated function.5Glycosyl residue in the parenthesis is branched off the first GalA.
Over the years, membrane-bound α1-4galacturonosyltransferase (GalAT) activity has been identified and partially characterized in mung bean10,11, tomato12, turnip12, sycamore13, tobacco suspension2, radish roots5, enriched Golgi from pea7, Azuki bean14, Petunia15, and Arabidopsis (see Table II). The pea GalAT was found to be localized to the Golgi7 with its catalytic site facing the lumenal side of the Golgi7. These results provided the first direct enzymatic evidence that the synthesis of HGA occurs in the Golgi. In in vitro reactions, GalAT adds [14C]GalA from UDP-[14C]GalA1,60 onto endogenous acceptors in microsomal membrane preparations to produce radiolabeled products of large molecular mass (i.e. ˜105 kd in tobacco microsomal membranes2 and ≧500 kd in pea Golgi7). The cleavage of up to 89% of the radiolabeled product into GalA, digalacturonic acid (diGalA) and trigalacturonic acid (triGalA) following exhaustive hydrolysis with a purified endopolygalacturonase confirmed that the product synthesized by tobacco GalAT was largely HGA. Thus, the crude enzyme catalyzes the reaction in vitro: UDP-GalAT+HGA(n)→HGA(n+1)+UDP. The product produced in vitro in tobacco microsomes was ˜50% esterified2 while the product produce in pea Golgi did not appear to be heavily esterified7. These results suggest that the degree of methyl esterification of newly synthesized HGA may be species specific and that methylesterification occurs after the synthesis of at least a short stretch of HGA. GalAT in detergent-permeabilized microsomes from azuki bean seedlings added [14C]GalA from UDP-[14C]GalA onto acid-soluble polygalacturonate (PGA) exogenous acceptors14. Treatment of the radiolabeled product with a purified fungal endopolygalacturonase yielded GalA and diGalA, confirming that the activity identified was a GalAT comparable to that studied in tobacco and pea. The azuki bean enzyme had a surprisingly high specific activity of 1300-2000 pmol mg−1 min−1, especially considering the large amount (3.1-4.1 nmol mg−1 min−1) of polygalacturonase activity that was also present in the microsomal preparations. As with the product made by tobacco, no evidence for the processive transfer of galactosyluronic acid residues onto the acceptor was obtained (see below).
TABLE IIComparison of apparent catalytic constants and pH optimum ofHGA-α1,4-galacturonosyltransferases1,2ApparentVmaxKm for(pmolPlantUDP-GalApHmg−1Enzyme2Source(μM)optimummin−1)RefGalAT1mung bean1.76.0~470010GalATmung beann.d.n.d.n.d.61GalATpean.d.56.0n.d.62GalATpean.d.n.d.n.d.7GalATsycamore770n.d.?13GalATtobacco8.97.81502GalAT (sol)3tobacco376.3-7.82903GalAT (sol)3Petunia1707.048015GalAT (per)4Azuki bean1406.8-7.82700141Adapted from ref 6.2Unless indicated, all enzymes are measured in particulate preparations.3(sol): detergent-solubilized enzyme.4(per): detergent-permeabilized enzyme.5n.d.: not determined.
GalAT can be solubilized from membranes with detergent3. Solubilized GalAT adds GalA onto the non-reducing end4 of exogenous HGA (oligogalacturonide; OGA) acceptors of a degree of polymerization of at least ten2. The bulk of the HGA elongated in vitro by solubilized GalAT from tobacco membranes3, or detergent-permeabilized Golgi from pea7, at roughly equimolar UDP-GalA:acceptor concentrations is elongated by a single GalA residue. These results suggest that solubilized GalAT in vitro acts nonprocessively, (i.e. distributively). The apparent lack of in vitro processivity of GalAT was recently confirmed by Akita et al. who, using pyridylaminated oligogalacturonates as substrates and high concentrations of UDP-GalA, showed that although OGAs can be elongated in a “successive” fashion with up to 10 GalA residues by solubilized enzyme from petunia pollen15, the kinetics of this response suggest a distributive mode of action. We have two working hypotheses as to why GalAT in vitro does not appear to act processively. One hypothesis is that the solubilized enzyme or the enzyme in particulate preparations does not have the required factors, or is not present in the required complex, to act processively. An alternative hypothesis is that for a Golgi-localized enzyme that synthesizes a complex polymer in a confined internal cellular compartment, such as GalAT, with sufficiently high concentrations of substrate, it would not necessarily be advantageous for the enzyme to act processively. In fact, the reaction velocity could be hindered under such conditions if the enzyme were processive65.
The apparent kinetic constants and pH optimum for the characterized GalATs are shown in Table II. We have performed additional kinetic studies in tobacco and radish that suggest that solubilized and membrane bound GalAT may have unusual apparent biphasic kinetics. We tested Vo for radish GalAT at 2 μM to 80 mM UDP-GalA and obtained a biphasic curve (FIG. 4), suggesting that the kinetics of GalAT, at least in the membrane and soluble fractions, are complex. Comparable results were also obtained for the solubilized radish and tobacco enzyme. The initial Vo vs [UDP-GalA] curve was hyperbolic and appeared to reach an initial maximum Vo of ˜300 pmol mg−1 min−1 at ˜1 mM UDP-GalA, confirming previous results reported for tobacco2,3. However, at ≧2 mM UDP-GalA there was a second hyperbolic increase in GalAT activity that reached a maximum of ˜2-4 nmol min−1 mg−1 with ˜20 mM UDP-GalA. In crude enzyme preparations it was not possible to determine the basis for the unusual kinetics. One possibility is that two GalATs were present, one with a low Km and one with a high Km. Another possibility is that UDP-GalA is both a substrate and an allosteric regulator of GalAT. Alternatively, a more “trivial” explanation is that at low substrate concentrations the kinetics of GalAT were effected by a catabolic enzyme (e.g. a phosphodiesterase) in the enzyme preparation.
As a first step towards elucidating the role of galacturonosyltransferase (GALAT) in pectin synthesis, the inventors herein identified an Arabidopsis gene encoding alpha1,4-galacturonosyltransferase 1 (GALAT1). The database searches using the amino acid sequence of the GALAT1 identified fourteen additional GALAT family members and ten GALAT-like genes. The identification of these genes and the availability of the sequence information allow the characterization of the enzyme, the use of these genes to produce mutated enzymes in vivo and in vitro, and transgenic plants producing modified pectins, and studies of the role of a specific GalAT in pectin synthesis. The advantages of the present invention will become apparent in the following description.